Insulin is a hormone that plays an important role in the metabolic regulation of glucose, an energy source for living organisms. Produced in pancreatic Langerhans β cells, insulin acts on cells carrying insulin receptors and promotes the uptake of glucose by these cells. The blood-sugar level in the body is maintained within an appropriate range by the function of insulin. Diabetes is one of the pathological conditions caused by insufficient insulin function due to some cause.
Major causes of insufficient insulin function include abnormal insulin secretion and decreased sensitivity to insulin. The former is called type 1 diabetes mellitus. Since responsiveness to insulin is maintained in type 1 diabetes mellitus, blood sugar level can be controlled by administering insulin. Type 1 diabetes mellitus is also called insulin-dependent diabetes mellitus (IDDM), and is the main cause of juvenile diabetes.
On the other hand, the latter is called type 2 diabetes mellitus. Type 2 diabetes mellitus is also called non-insulin dependent diabetes mellitus (NIDDM), and is the type of diabetes frequently found in adults. In Japan, 95% of diabetes patients are said to have type 2 diabetes mellitus. Since the body's responsiveness to insulin is decreased in these patients, even an insulin administration cannot regulate the blood sugar level. Type 2 diabetes mellitus is thought to develop due to several genetic defects and environmental factors such as obesity, stress, and aging. At present, approximately 7,400,000 type 2 diabetes patients are said to exist in Japan, and the number is increasing with the aging of the population. The number of patients is even predicted to be as many as 16,200,000, when including prediabetes patients. Therefore, the diagnosis and treatment of type 2 diabetes mellitus is an important research issue for the modern society.
To date, the causative gene of type 2 diabetes mellitus has not been revealed. Presumed candidate genes are genes of factors involved in the mechanism of insulin action, or genes of factors involved in insulin secretion. Factors thought to be involved in insulin action are:                insulin receptor,        insulin receptor substrate-1 (IRS-1),        glucose transporter type 4, etc.        
Genes of factors predicted to be involved in insulin secretion are:                glucose transporter type 2,        glucokinase,        mitochondrial genes, etc.        
For insulin to act on a target cell, it must bind to the insulin receptor present on the target cell membrane. Furthermore, there are many reports of insulin resistance in the early stage of type 2 diabetes (Non-Patent Document 1/Taylor, S. I. Diabetes 41:1473-1490, 1992). In view of these facts, the relationship between insulin receptor abnormalities and diabetes has also been examined. If abnormalities are present in insulin receptor function, strong insulin resistance will arise, resulting in severe diabetes.
Recently, many insulin receptor abnormalities have been discovered by researchers including the present inventors, and it is becoming evident that test results and symptoms of patients vary depending on the type of mutation (Non-Patent Document 2/M. Taira et al., Science 245:63-66, 1989; Non-Patent Document 3/F. Shimada et al., Lancet. 335:1179-1181, 1990). This suggests that a part of the pathogenesis of type 2 diabetes mellitus may be defects in the insulin receptor gene. The present inventors have actually identified one of the polymorphisms that allow genetic diagnosis of type 2 diabetes mellitus, and have already filed a patent application (Patent Document 1/Unexamined Published Japanese Patent Application No. (JP-A) Hei 8-103280).
Insulin receptors are heterotetrameric receptor proteins composed of two subunits, αand β. The α-subunit is present outside the cell and the β-subunit penetrates the cell membrane. The α-subunit is linked to the extracellular domain of the β-subunit via an SS bond through an SH group in a cysteine residue on the C-terminal side thereof.
When insulin binds to the α-subunit, a tyrosine residue in the intracellular domain of the β-subunit is autophosphorylated, and the insulin signal is transmitted to the cell. After binding with insulin, the insulin receptor present in the cell membrane is then taken into the cell by endocytosis (the half-life of the receptor is seven hours). The number of insulin receptors decrease with the increase of insulin concentration. This is called down regulation.
In the polymorphism of insulin receptor found by the present inventors, Thr at position 831 in the β-subunit is mutated to Ala (IRA831). Insulin receptor dysfunction caused by this amino acid substitution has not been confirmed. However, genetic statistics indicated that there is a strong relation between IRA831 and type 2 diabetes mellitus.
Disorders due to receptor abnormalities or the presence of free receptors in blood have recently been reported for some diseases (Non-Patent Document 4/Frode, T. S., Tenconi, P., Debiasi, M. R., Medeiros, Y. S., “Tumour necrosis factor-alpha, interleukin-2 soluble receptor and different inflammatory parameters in patients with rheumatoid arthritis.” Mediators Inflamm. 2002 Dec; 11(6): 345-9; Non-Patent Document 5/Baron, A. T., Cora, E. M., Lafky, J. M., Boardman, C. H., Buenafe, M. C., Rademaker, A., Liu, D., Fishman, D. A., Podratz, K. C., Maihle, N. J., “Soluble Epidermal Growth Factor Receptor (sEGFR/sErbB1) as a potential Risk, Screening, and Diagnostic Serum Biomarker of Epithelial Ovarian Cancer.” Cancer Epidemiol Biomarkers Prev., 2003 Feb; 12(2): 103-13; Non-Patent Document 6/Beguin, Y “Soluble transferrin receptor for the evaluation of erythropoiesis and iron status.” Clin. Chem. Acta., 2003 Mar.; 329(1-2): 9-22). Furthermore, hyperglycemia and hyperinsulinemia have been observed in transgenic mice that release the insulin receptor α-subunit into the blood (Non-Patent Document 7/ERIK M. SCHAEFER et al. DIABETES vol. 43, 143-153; 1994). However, in humans, the presence of free insulin receptors in blood has not been reported.
Substances whose levels in biological samples change with disease states are often useful as diagnostic markers for the diseases. For example, substances in biological samples that can be used as cancer indicators are called tumor markers. When a cancer is present, tumor marker levels in these biological samples change significantly compared to those in healthy subjects. Therefore, based on the measured value of tumor markers, it is possible to estimate the possibility of cancer in the subject. Usually, definite diagnosis of the presence or absence of cancer using only tumor markers is considered difficult. However, measurement of tumor markers is considered effective in screening for test subjects who require more advanced and detailed cancer tests.
Among tumor markers, there are some whose measured levels change in correlation with the size and progression of the cancer. Such tumor markers are useful as indicators for observing therapeutic effects on cancer.
Many tumor markers have been reported so far. Generally, tumor markers are often substances that originally exist in normal tissues. Even in healthy people, the measured level of tumor markers may change due to physiological conditions and diseases other than cancer. Therefore, subjects who do not have cancer may be judged as positive, i.e., “false-positive”. Conversely, tumor markers of subjects who should be diagnosed as having cancer may remain within a normal range. In this case, subjects who should be positive are determined as negative, i.e., “false-negative”.
Positive or negative judgments are made based on the relationship between the measured tumor marker levels and the cutoff values. More specifically, when using a tumor marker with a measured value that is significantly high in cancer patients, cancer is suspected if the measured value is greater than or equal to a certain value (cutoff value). Generally, if a cutoff value is set high, false-negative will increase and false-positives will decrease. Conversely, lowering the cutoff value decreases false-negatives, but increases false-positives. Since the increase in false-negatives means that some cancers would go undetected, keeping it to a minimum is preferable. On the other hand, increase in false-positives will lead to advanced tests on subjects who do not need such. Therefore, tumor markers that can keep false-negatives and false-positives within an acceptable range are considered more practical.
Permissible false positives and false negatives vary depending on the level of difficulty of the diagnosis, presence or absence of a therapeutic method, or the number of patients of a cancer. In addition, an important criterion is whether or not other tumor markers that should be used for comparison are known. When confirming therapeutic effects or monitoring recurrence in patients known to have cancer, tumor markers are continuously monitored. In such applications, the response characteristics of the marker to cancer are given more weight than the issue of false negativeness or false positiveness. Based on such criteria, several tumor markers have been put to practical use. Examples of currently widely-used tumor markers are as follows:
AFP (liver cancer, kidney cancer, cancer of the digestive system)
CEA (liver cancer, kidney cancer, cancer of the digestive system)
CA19-9 (pancreatic cancer, biliary tract cancer, colon cancer)
CA125 (ovarian cancer)
PSA (prostate cancer)
NSE (small-cell lung cancer)
CYFRA (lung cancer —squamous cell carcinoma—)
Some of these tumor markers have been practically applied for specific types of cancers, and others have been recognized as tumor markers for a relatively wide variety of cancers. For example, NSE, CYFRA, or such are tumor markers for specific types of cancers. On the other hand, AFP, CEA, or such are tumor markers that are positive in a relatively wide variety of cancers.
Tumor markers having low specificity to cancer types and which are applicable as tumor markers for a variety of cancers are particularly referred to as “broad-spectrum tumor markers”. Broad-spectrum tumor markers are more advantageous than markers specific to a certain cancer type in that they can be utilized to detect or assess therapeutic effects on a broad range of cancer types. The usability of known broad-spectrum tumor markers such as AFP or CEA as tumor markers has been acknowledged for certain cancer types such as cancers of the liver, digestive system, and kidney. However, their usefulness as tumor markers is not always recognized in other cancer types. Therefore, provision of tumor markers that can cover cancer types that are difficult to diagnose with known tumor markers would be useful.
As mentioned above, presence of free receptors in blood has been reported in some diseases (Non-Patent Documents 4-6). Transgenic mice that release the insulin receptor α-subunit into the blood have also been confirmed to exhibit hyperglycemia or hyperinsulinaemia (Non-Patent Document 7). However, in humans, there are no reports that suggest a relationship between cancer and free insulin receptors in blood. The finding that correlated the insulin receptor α-subunit to cancer is the work of a group at Genentech and Memorial Sloan Kettering Cancer Center in the United States, in February 1985, who cloned the human insulin receptor gene, determined the complete amino acid sequence, and analyzed the amino acid sequence to reveal homologies with the epidermal growth factor (EGF) receptor and the protein of cancer gene src.